Extraction of penicillin



April 4, 1950 G. J. PIEROTTI 2,503,214

EXTRACTION 0F PENICILLIN Filed Feb. 14, 1945 Seled'ive Il Feed AcdRecover Soweni' 2| Sowenf 4 F3 Exi'mm` Raffinaie Aqueous Rafflnaie welHamm Soveni Recovrg Selec've Exi'md olvenl' 7 Soweni Aqueous Raf'inne Buhis Ahorneg L M Patented Apr. 4, 1950 ax'raAc'noN or rnNlclLLm Gino J.Pieroni. albany, celu., mignon zo sneu Development Company, SanFrancisco, Calif a corporation of Delaware Application February 14,1945, Serial No. 577,824

14 Claims. (Cl. 26o-302) This invention involves a process for therecovery and puriiication of antibiotics, such as penicillin. Moreuparticularly, it relates to a process for the separation-of organicimpurities, more soluble in acidic water than is penicillin, from asolution containing' theml and the penicillin. This invention lisalcontinuation-impart of application, Serial No. V540,922,1iled June 19,1944 now abandoned, and particularly relates to an improvement in StepVI therein for the removal of acids stronger than penicillin.

Anitbiotic substances such as penicillin are the products of certainfungus growths. There are a number of antibiotics known, but onlypenicillin is being commercially raised today, although it is quitelikely that others may be produced in the near future. For a discussionof sources of antibiotics, and particularly of penicillin, refer toEndeavor" (Kynock Press, London for I. C. I.) vol. 3, No. 9, January1944. pp. 3 to 14.

Penicillin is an organic acid, a basteriostatic agent and an importantdrug for combating diseases and for preventing the spread of infections.It is produced during the growth of certain molds. such as penicilliumnotatum, in an aqueous solution. There are several different ways ofgrowing these molds, for example, in submerged liquid cultures. or onliquid surface cultures, or on moist bran. The aqueous broth obtainedfrom the various cultures usually contains between about .01 and .001%by weight of penicillin or between about and 200 Oxford units per ml.(see the article by Floury and Jennings in the British Journal ofExperimental Pathology, vol. 23, page 120, of June 1942. The broth alsocontains varying amounts of solids, non-ionic impurities, amphotericmaterials, diiferent organic impurities including acids both strongerand weaker than penicillin, etc. There are several different penicillinsknown, for example: penicillins F and G, allo penicillin, etc., some ofwhich have different solubilities in different solvents.

It is an object of this invention to improve both the purity and therecovery of antibiotics, particularly penicillin. Another purpose is tocarry out this purification and recovery in a minimum space of time.Another purpose is to effectively and eiliciently remove organicimpurities normally associated with penicillin, including acids strongerthan penicillin, from aqueous solutions containing them and penicillin.Still another purpose is to achieve this by a continuous liquidliquidcountercurrent solvent extraction process.

Generally speaking, this invention comprises 2 separating penicillinfrom water-soluble impurities normally associated therewith in an acidicaqueous solution of penicillin by countercurrently extracting saidsolution with a liquid solvent phase which has the property ofdecreasing in solvent power for penicillin and increasing in selectivityfor penicillin in the direction of the flow of the solvent; orconversely, extracting said aqueous solution with a liquid solvent underconditions wherein the solvent phase possesses an increasing solventpower and a diminishing selectivity for penicillin in the direction ofiiow of said aqueous phase. For example, an aqueous solution ofpenicillin first is contacted with a recovery solvent (as defined later)to extract substantially all the penicillin, as well as some non mallyassociated impurities, from the aqueous phase, and thereafter a solventmore selective for penicillin miscible with the recovery solvent isadded to form a mixed solvent phase and to spring at least some of theseimpurities from the mixed solvent phase. The resulting solvent phase iswashed with an aqueous solution to insure the removal of the sprungimpurities. The resulting two phases, one a solvent phase containingpenicillin and the other an acidic aqueous phase containing theimpurities. are separately removed at opposite ends of the extractionzone. It is usually desirable to remove the penicillin from the solventphase and re-use the solvents therein. Thismay be accomplished by anysuitable means, such as by neutralization of the penicillin acid with abasic material such as calcium, strontium or barium hydroxide, sodium orpotassium bicarbonate, phosphate, stearate, tartrate, etc. Thepenicillin removed from the solvent phase may be further purified andconcentrated before it is ready for storage or for use. Before treatingthe raw penicillin solution or broth by the process of this inventionthe solids therein should be removed. It is preferred, but notessential, that some of the soluble and liquid impurities be removedalso. and/or the penicillin be concentrated from the raw broth prior totreating the penicillin solution by the process of this invention. Thesepre-treating steps may be accomplished in diierent ways, for example: byadsorption of the penicillin on charcoal and elution of the absorbate inan aqueous solvent (see Chemical and Metallurgical Engineering April1944, pages 94-98 and 13D-133), or by a liquid extraction process suchas that shown in Pierotti et al co-pending application, Serial No.540,922, tiled June 19. 1944. It is desirable that the penicillin in thesolution fed to the extraction 3 process of this invention have a.concentration above about .001% by weight.

Penicillinacid decomposes very easily at temperatures slightly above C.Strong acids. strong bases, many heavy metals and certain otherre-agents destroy it. Accordingly, this process is carried out asrapidly as possible and as closely to 0 C. as possible without freezingthe solutions involved therein. All acids or bases introduced to adjustthe hydrogen ion concentration in the aqueous phase in the process areadded in dilute aqueous solutions.

No one solvent is known which has both high solvent power and highselectivity for penicillin. Therefore, both a high yield and a highpurity cannot be obtained in an extraction of reasonable efficiency withonly one solvent. This invention employs the use of at least two organicsolvents, one of which has high solvent power for penicillin(hereinafter called the recovery solvent") and another of which has highselectivity for penicillin (hereinafter called the selective solvent).The recovery solvent has a higher distribution ratio K for penicillinthan does the selective solvent.

whereas the selective solvent has a higher alpha" value for penicillinwith respect to the impurities normally associated therewith than thatof the recovery solvent.

K concentration of penicillin in rccoverv solvi concentration ofpenicillin in the aqueous stage K of penicillin inHrecg/ery solv ci \tBoth K and a must be greater than one, and the greater the differencesbetween the two alpha values as well as between the two K values of thechosen solvents the better the recovery and the purer the extractedpenicillin.

The recovery and selective solvents in admixture with water must formtwo separate phases. It is desirable that they should be less than andpreferably less than 1 by volume soluble in water. 'I'hey should bemiscible with each other and may have different polarities. They mustnot react with the penicillin or cause it to decompose and must beliquid and not too viscous at temperatures as low as -5 C. They shouldhave a melting point below 5 C. Their solvent char acteristics must notbe materially affected by the compounds in the penicillin solutionincluding the penicillin itself. Their densities must differ from thatof water and may vary from each other, that is, one may be more densethan water and the other may be less dense than water. In order toreadily form a separate liquid phase with water it is desirable that thedensity of the solvent differ from that of water by at least about Eachsolvent may be a mixture of individual solvents.

Some suitable organic recovery solvents are formates, acetates,propionates, butyrates, etc., containing from 4 to 8 carbon atoms suchas: n-

and iso-propyl acetate, nand iso-butyl acetate,

amyl acetates, ethyl and propyl chloro acetates; lactates containingfrom 5 to 10 carbon atoms; dimethyl phthalate; chloroform; ethylenedichloride; diethylene ether: di-isopropyl ether; methyl propyl ketone,methyl iso-butyl ketone, isophorone; etc.; or mixtures of one or more ofthe above. In order to have the proper degree of polarity, the recoverysolvents should be at least .1% and preferably at least .2% by volumesoluble in water.

Some suitable organic selective solvents are light hydrocarbons such as:isoand n-butanes, isoand n-pentanes, isoand n-hexanes, heptanes.

benzene, toluene, etc.; and chlorinated hydrocarbons such as:chloroform, carbon tetrachloride, ethylene dichloride, etc. Solvents oflow tendency to form hydrogen bonds are more selective for penicillinthan those which have a high tendency to form hydrogen bonds and produceassociated liquids. Thus, generally speaking, the relatively lighthydrocarbons and halogenated derivatives thereof, being more or lessnon-polar organic solvents are better selective solvents than recoverysolvents, although some halogenated hydrocarbons may be used as recoverysolvents in combination with still less polar solvents.

Suitable recovery and selective solvent combinations are: chloroform andiso-pentane. methyl iso-butyl ketone and iso-pentane, methyl iso-butylketone and chloroform, etc. The choice of a combination depends upon theselective and recovery characteristics of each solvent relative to thedif ferent forms of penicillin which may have to be extracted. Onesolvent may be the recovery solvent for one form of penicillin while thesame solvent may be the selective solvent for another form ofpenicillin.

An aqueous phase is necessary throughout the extraction zone. Tomaintain, the solvent extract may be washed with water by adding waternear the solvent outlet of said zone. Moreover, water may be added atone or more other points throughout the extraction zone. This water maybe pure or distilled water, or it may contain a small amount of anelectrolyte such as an acid or a buffer substance which may have severallevels of ionization, in order to maintain the pH of the aqueous phasethroughout the extraction zone between about 1.5 and 5andpreferablylbetween about 2 and 3. Suitable acids are dilute sulfuric,phosphoric, etc., acids. If desired, the electrolyte may be added to theextraction zone with the feed containing the penicillin. The functionand control of such aqueous solutions containing ions is fully describedin Pierotti et al. co-pending application, Serial No. 540,923, filedJune 19, 1944 now abandoned.

The drawing discloses schematic diagrams of three possible flows whichmay be used in carrying out the process of this invention (for therecovery and purification of a solution of penicillin). Figure I shows asingle continuous extraction column which may be used when both solventsare less dense than the aqueous phase. Figure II represents a flowdiagram comprising two continuous liquid-liquid extraction columns whichmay be used when the recovery solvent isl less dense and the selectivesolvent is more dense than the aqueous phase. Figure III is a flowdiagram of a continuous or batch liquid-liquid extraction apparatuscomprising a series of mixers and settlers.

Referring to Figure I, the countercurrent extraction zone comprising asingle column l0 is provided with a feed inlet line l l for thepenicillin solution intermediate the ends of the column, a solventextract outlet I2 at the top of the co1- umn and an aqueous ramnateoutlet I3 at the bottom of the column. The penicillin solution isusually an aqueous solution, although it may be a solution in adifferent solvent if a suitable aqueous phase is maintained throughoutthe extraction zone. The recovery solvent is introduced into the columnl0 through line Il near the aqueous ramnate outlet in order to insurethe maximum recovery of penicillin from the aqueous phase even to theextraction of some of its closely related impurities, if necessary,before the aqueous phase is withdrawn and discarded through line I3.Water, or a suitable aqueous solution containing a buffer and preferablyhaving a pH of about 7, is introduced through the valved line I5 nearthe solvent phase outlet in order to maintain an aqueous phasethroughout the column III and secure the highest selectivity forpenicillin at the solvent outlet. The selective solvent is introducedinto the column through line or lines I6 'at one or several pointsbefore or after the feed contacts the 'recovery solvent, or with thefeed. Dilute acid, as well as buffer and/or dilute base, 'may beintroduced into the feed -line II through line I1 or at any other pointor points (not shown) along the column to maintain the pH of the aqueousphase within the column below aboutv5. It is desirable to maintain anincreasing pH gradient in the aqueous phase, in that section`ofV thecolumn or extraction zone between the feed and'water inlets Il and I5,respectively, and to maintain more or less of a constant pH in theaqueous phase in that section of the extraction zone between therecovery solventA and feed inlets I 4 and I I, respectively.

In column I0, the penicillinis extracted from the aqueous phase into thesolvent phase as it passes down the column. The selective solvent tendsto spring the impurities (extracted with the penicillin by the recoverysolvent) from the solvent phase into the aqueous phase.y The waterintroduced through line I5 washes the impurities and may also wash someof the penicillin from the solvent phase, which penicillin is laterre-extracted from the aqueous phase as it passes down the column. Thisproduces a reflux of penicillin in the upper part of the column.

Figure II discloses a modification of the process shown in Figure I,wherein the selective solvent, such as chloroform, is more dense thanthe aqueous phase, and the recovery solvent, such as methyl iso-butylketone, is less dense than the aqueous phase. In order to maintain acontinuous process the liquid-liquid extraction zone must be dividedinto two columns and 30; column 20 for the recovery solvent and aqueousphase feed,

and column for both solvents and water Wash*v aqueous phase. The lessdense recovery solvent is introduced near the bottom of column 20through line 2i near the aqueous raiiinate outlet 22 and the penicillinsolution is fed into the column 20 near its top through line 23. Arecovery solvent phase containing penicillin is withdrawn through line24 at the top of column 20 for transfer to column 30. The top of column30 is provided with a top aqueous phase outlet line 3| for transfer ofthe aqueous phase containing impurities to the top of column 20. Thedense selective solvent from line 32 is introduced into the recoverysolvent phase from the top of column 20 into the transfer line 24through line 33, in a quantity at least sufficient to produce a mixedsolvent phase denser than the aqueous phase present in column 30.Additional selective solvent may be added to other points along column30, if desired, such as through valve line 34. Water or an aqueous buersolution is introduced near the bottom of column 3U through line 35 nearthe mixed solvent outlet line 36. Acid, base, and buffer may likewise beadded to the feed through line 24 and/or at different points (not shown)along columns 20 and 30 as desired.

Figure III comprises two mixer-settler units, which is the minimumnumber of units necessary for the process of this invention, involvingtwo different solvents. However, additional mixersettler units may beused in series to obtain any desired number of extraction stages. InFigure III, the penicillin solution, which may be previously acidiiiedby dilute acid introduced through `line 48, is fed through line 4I intomixer 4l wherein it is mixed with the recovery solvent introducedthrough line 42. The aqueous raillnate from a following settler may alsobe introduced into mixer through line 43 to insure complete recovery ofany penicillin from said ramnate before it is discarded. The resultingmixture oi.' recovery solvent and aqueous phase is passed from mixer 40through line 44 into settler 45, wherein the aqueous phase is allowed tosettle and is withlowed to settle and is intermittently (or continu- Illously) withdrawn through line 53 for discard through line 51, or forreturn to mixer 40 through line 43. covered and` purified through topline 58.` l

If the density of the solvents mentioned in describing the above figuresare reversed with re'- spect to water, the columns and settlers shouldbe inverted.

Columns I0, 20 and/or 30 may be of any conventional design with packing,baille plates, or the like, or may, and preferably, be rotary extractioncolumns. The drawing does not shown auxiliary pumps, pipes, valves,outlets, inlets, tanks, heating'and cooling lines, solvent recoverysystems, pH indicators and controllers, acid, base, and butler injectionlines, etc., which may be necessary in carrying out the process, becausethe proper placement of this equipment will be evident to one skilled inthe art.

The amounts of each solvent, recovery and selective, employed dependsupon various factors including: their relative a and K values; theirrelative densities; their relative rates of transfer of impurities; theratio of the various forms of penicillin in the penicillin solutionfeed; the properties, purity and yield of the final products; etc.However, generally speaking, the ratio of solvent phase to aqueous phasein the extraction zone varies between about .1:1 and 10:1 by volume, andpreferably about 1:1; and the ratio of recovery solvent to selectivesolvent varies between about .05:1 and 20:1 by volume, and preferablybetween about .l :1 and 10:1.

The following examples illustrate the eil'ectiveness of the process ofthis invention.

Example I An aqueous penicillin solution having a purity of about Oxfordunits per mg. of dissolved solids and containing naturally associatedimpurities was fed into a liquid-liquid rotary extraction column,similar to column I0 in Figure I, at a rate of 15.9 cc. per minutethrough line II together with 1 cc. per minute of a 3% by weight aqueousphosphoric acid solution through line Il to produce a feed having a pHof 2.0. 15.0 cc. per minute of methyl iso-butyl ketone was introducedthrough line I4; 7.5 cc. per minute of water was introduced through lineI5; and varying amounts of iso-pentane were introduced f through line I6 directly opposite the line II.

The solvent phase containing 'there-f penicillin is withdrawn 7 154.0cc. per minute of solvent extract were withdrawn from the top line I2and 24.4 cc. per minute of aqueous raffinate were discarded throughbottom line I3. The purity of the penicillin removed from each solventextract by an aqueous solution having a pH of 6.8 are as follows:

Percent Methyl hasta lsogysglvlggom Pass; gf tristan n cc./minuto FfmmtPenicillin Units per mg.

(l 10() 90. 5 165 2 R8 90. (l 209 4 79 90. 0 237 lll l (i l 245 Theseresults show that the purity of penicillin in the final product issubstantially increased by the addition of the selective solventiso-pentane to the recovery solvent methyl iso-butyl ketone.

Example II An aqueous solution of penicillin having a purity of 100Oxford units per mg. of dissolved solids and containing dissolvedimpurities was fed into a rotary extraction column at a rate of 16 cc.per minute together with .9 cc. per minute of 3% solution of phosphoricacid to produce a solution having a pH of 2.0. This aqueous feed wascontinuously countercurrently contacted with a mixed recovery solvent(5U-50% by volume chloroform and methyl iso-butyl ketone) having adensity greater than that of water and which solvent was introduced nearthe top of the column at the rate of 15 cc. per minute. A lean aqueousramnate was withdrawn from the top of the column at a rate of 24 cc. perminute. The aqueous solvent extract phase withdrawn from the bottom ofthe column was introduced near the top of a second rotary extractioncolumn. l5 cc. per minute of more chloroform, as a selective solvent,was introduced into the solvent line between the first and secondextraction columns and 7.5 cc. per minute of water was introduced nearthe bottom of the second extraction column. A rich solvent extract waswithdrawn from the bottom of the second column at a rate of 30 cc. perminute which was recovered and tested and found to contain '71% of thepenicillin in the feed having a purity of 382 Oxford units per mg. ofdissolved solids. in comparison with a recovery of 67% and a purity of283 Oxford units per mg. in the same apparatus with the same feed butwithout the addition of the extra 15 cc. of chloroform as a selectivesolvent.

Example III 16.2 cc. per minute of an aqueous solution of penicillinhaving a purity of about 100 Oxford units per mg. of dissolved solidsand containing impurities was mixed with 1.5 cc. per minute of 3%phosphoric acid through line 24 to produce a solution having a pH of2.2. This latter solution was introduced near the top of a rotary'liquid-liquid extraction column, similar to column 20 in Figure II,through line 23 and continuously countercurrently extracted with 15 cc.per minute of methyl iso-butyl ketone introduced through line 2|.Aqueous-raillnate at the rate of 25.2 cc. per minute was withdrawnthrough line 22 and was tested to contain 1.8% of the penicillin in thefeed. The methyl iso-butyl ketone solvent solution was withdrawn throughline 24, mixed with 15 cc. per minute of chloroform introduced throughline Il and passed into column 30 where it was contacted with '7.5 cc.per minute oi puro water introduced through line 35. Rich solventextract containing penicillin was withdrawn through line 3B at the rateof 30 cc. per minute and was tested to contain 95.3% of the penicillinin the original feed. The sodium salt of this penicillin having a pH of6.8 had a purity of 434 Oxford units per mg. The aqueous solution fromthe top of column 30 was passed back into column 20 through line 3l, asshown in Figure 1I.

Example IV 150 cc. of methyl iso-butyl ketone solvent extract containingpenicillin and impurities was mixed with 15 cc. of chloroform and a 150cc. of pure water and allowed to settle (see mixer and settler 50 and 55in Figure III). The sodium salt of penicillin recovered from theresulting solvent phase showed a recovery of of the original penicillinin the methyl iso-butyl ketone solvent extract solution having a purityof 570 Oxford units per mg. of dissolved solids, while the resultingaqueous phase was tested to contain 10% 0l.' the original penicillinhaving a purity of 84 Oxford units per mg. of dissolved solids.

Another 150 cc. sample of methyl iso-butyl ketone of the same solventextract of penicillin was washed with only 75 cc. of water and allowedto settle. The sodium salt of penicillin recovered from the resultingsolvent phase had a purity of only 360 Oxford units per mg. of dissolvedsolids.

Example V cc. of a 90-10% methyl iso-butyl ketonechloroform mixedrecovery solvent extract containing penicillin and impurities was mixedwith 100 cc. more of chloroform and 100 cc. of water and allowed tosettle. The sodium salt of penicillin prepared from the resultingaqueous phase was tested to contain 9.4% of the original penicillinhavlng a purity of Oxford units per mg. of dissolved solids. The sodiumsalt prepared from the resulting solvent phase was tested to contain90.6% of the original penicillin having a purity of 450 Oxford units permg. of dissolved solids.

Another 100 cc. sample of the same original methyl iso-butylketone-chloroform solution containg penicillin was Washed with only 50cc. of water and allowed to settle. The sodium salt of penicillinprepared from the resulting solvent phase was tested to have only apurity of 347 Oxford units per mg. of dissolved solids.

The invention claimed is:

1. In a process for extracting penicillin from an aqueous solutioncontaining it and impurities naturally associated therewith, the stepscomprising contacting said aqueous solution ilrst with a solvent phasecomprising essentially a substantially water-insoluble. polar organicrecovery solvent and a substantially water-insoluble, substantiallynon-polar organic selective solvent to form a contacted aqueous solutionand solvent phase, separating said phases, and thereafter furthercontacting said contacted aqueous solution with a solvent comprisingessentially the recovery solvent only, said rccovery solvent having ahigher solvent power for penicillin than water, said selective solventhaving a higher selectivity for penicillin than said recovery solvent,and maintaining in said aqueous solution during said contacting a pHbetween about 1.5 and 5.

2. The process of claim 1, wherein the ratio of recovery solvent toselective solvent in said mixed solvent phase is between about .05:1 and20:1 by volume.

3. The process of claim 1, wherein said recovery solvent and saidselective solvent are less than v. soluble in water and said recoverysolvent is more than .1% v. soluble in water.

j 4. 'Ihe process of claim l, wherein said recovery solvent comprisesessentially methyl iso-butyl ketone. Y l

5. The process of claim 1, wherein said selective solvent comprisesessentially chloroform.

6. The process of claim 1, wherein said selective solvent comprisesessentially iso-pentane.

7. A process for, extracting penicillin from an aqueous solutioncontaining it and impurities naturally associated with penicillin,comprising treating said aqueous penicillin solution with an organic,substantially water-insoluble, polar recovery solvent to produce anextract phase containing penicillin and at least some of said impuritiesand aqueous rafllnate phase, separating said extract and railinatcphases, treating said extract phase with an organic, substantiallywater-insoluble, substantially non-polar selective solvent and water toproduce a mixed solvent phase containing penicillin and an aqueous phasecontaining said impurities and separating said phases, said recoverysolvent having a higher solvent power for penicillin than water, saidselective solvent having a higher selectivity for penicillin than saidrecovery solvent, and maintaining in said aqueous phase a pH betweenabout 1.5 and 5.

8. A process for extracting penicillin from an aqueous solutioncontaining it and impurities naturally` associated with penicillin,comprising treating saidaqueous penicillin solution with an organic,substantially water insoluble, polar recovery solvent to produce anextract phase containing penicillin and at least some of said impuritiesand aqueous ramnate phase, separating said extract and railinate phases,treating said extract phase with an organic substantiallywaterinsoluble, substantially non-polar selective solvent and water toproduce a mixed solvent phase containing penicillin and an aqueous phasecontaining said impurities, separating said phases, said recoverysolvent having a higher solvent power for penicillin than water, saidselective solvent having a higher selectivity for penicillin than saidrecovery solvent, and recycling said aqueous phase to join with saidaqueous penicillin solution in the first treating step, and maintainingin said aqueous phase a pH between about 1.5 and 5.

9. In a process for extracting penicillin from an aqueous solutioncontaining it and impurities naturally associated with penicillin withorganic solvents whereby liquid and solvent and aqueous phases areproduced, the improvement comprising: maintaining in said aqueous phasea pH between about 1.5 and 5, introducing said aqueous solution into aliquid extraction zone intermediate the outlets of said aqueous andsolvent phases, introducing an organic, substantially water-insoluble,polar recovery solvent into said zone intermediate the inlet of saidaqueous solution and said aqueous phase outlet, introducing water intosaid zone intermediate the inlet of -said aqueous solution and saidsolvent phase outlet, and introducing an organic, substantiallywater-insoluble, substantially non-polar selective solvent intermediatethe inlets of said water and said recovery solvent, said recoverysolvent having a higher solvent power for penicillin than water and saidselective solvent having a higher selectivity for penicillin than saidrecovery solvent.

l0. The process of claim 9, wherein said se- 10 lective and recoverysolvents are miscible in each other throughout said extraction zone.

11. The process of claim 9, wherein said recovery solvent comprisesessentially chloroform and said selective solvent is a lighthydrocarbon.

12. In a process for the recovery and purification of penicillin from anaqueous solution containing penicillin and other impurities morewater-soluble than penicillin, naturally associated therewith, the stepscomprising: contacting said aqueous solution with an organic,substantially water-insoluble, polar recovery solvent having a highersolvent power for penicillin than water, to produce an aqueous phase anda solvent phase, adding to said solvent phase an organic, substantiallywater-insoluble, substantially non-polar selective solvent miscible withsaid solvent phase and having a higher selectivity for pencillin thansaid recovery solvent; and contacting the resulting solvent mixture withwater to produce 'an extract phase containing said solvents andpenicillin, and a rafllnate phase containing said water and some of saidimpurities.

13. In a process for the recovery and purification of penicillin from anaqueous solution containing penicillin and other impurities morewater-soluble than penicillin, naturally associated therewith, the stepscomprising: contacting said aqueous solution with an organic,substantially water-insoluble, polar recovery solvent having a highersolvent power for penicillin than water, to produce an aqueous phase anda solvent phase; adding to said solvent phase an organic, substantiallywater-insoluble, substantially non-polar selective solvent miscible withsaid solvent phase and having a higher selectivity for penicillin thansaid recovery solvent; and contacting the resulting solvent mixture withwater to produce an extract phase containing said solvents andpenicillin, and a raiilnate phase containing said water and some of saidimpurities and recycling said raflinate phase to the rst contact stepfor contact with said recovery solvent and maintaining in said aqueousphase a pH between about 1.5 and 5.

14. In a process for the recovery and purification of penicillin from anaqueous solution having a pH between 1.5 and 5 and containing otherimpurities more water-soluble than penicillin naturally associated withpenicillin, including acids stronger than penicillin, the stepscomprising: contacting said solution with an organic, substantiallyWater-insoluble, polar recovery solvent having a higher solvent powerfor penicillin and some of said stronger acids than water, to form twoliquid phases and to remove substantially all of said penicillintogether with part of said impurities from the aqueous to the solventphase; adding to said solvent phase an organic, substantiallywater-insoluble, substantially non-polar selective solvent miscible withsaid recovery solvent and having a higher selectivity for penicillinthan said recovery solvent, to retain said penicillin in the resultingmixed solvent phase; contacting said resulting mixed solvent phase withwater to wash out substantially all the impurities present therein, toproduce a mixed solvent extract phase containing substantially purepenicillin, and a rainate phase containing said impurities: andrecycling said railinate phase to the iirst contact step for contactwith said recovery solvent.

GINO J. PIEROTTI.

(References on following page) 11 12 REFERENCES CITED v OTHER REFERENCESThe following references are of record 1n the WinthropReports W. I. meof this pement: unset II, August 16, 1941. plies 177-189.

` UNITED STATES PATENTS Summary Report on Penicillin, March 29, 1944,

s pp. 1-4.

1. IN A PROCESS FOR EXTRACTING PENICILLIN FROM AN AQUEOUS SOLUTIONCONTAINING IT AND IMPURITIES NATURALLY ASSOCIATED THEREWITH, THE STEPSCOMPRISING CONTACTING SAID AQUEOUS SOLUTION FIRST WITH A SOLVENT PHASECOMPRISING ESSENTIALLY A SUBSTANTIALLY WATER-INSOLUBLE, POLAR ORGANICRECOVERY SOLVENT AND A SUBSTANTIALLY WATER-INSOLUBLE, SUBSTANTIALLYNON-POLAR ORGANIC SELECTIVE SOLVENT TO FORM A CONTACTED AQUEOUS SOLUTIONAND SOLVENT PHASE, SEPARATING SAID PHASES, AND THEREAFTER FURTHERCONTACTING SAID CONTACTED AQUEOUS SOLUTION WITH A SOLVENT COMPRISINGESSENTIALLY THE RECOVERY SOLVENT ONLY, SAID RECOVERY SOLVENT HAVING AHIGHER SOLVENT POWER FOR PENICILLIN THAN WATER, SAID SELECTIVE SOLVENTHAVING A HIGHER SELECTIVITY FOR PENICILLIN THAN SAID RECOVERY SOLVENT,AND MAINTAINING IN SAID AQUEOUS SOLUTION DURING SAID CONTACATING A PHBETWEEN ABOUT 1.5 AND 5.